Background: MicroRNAs (miRs) are altered in breast tumors, influencing tumor development and progression. As miRs are remarkably stable in circulation, they also have enormous potential as non-invasive biomarkers for breast cancer. Indeed, circulating miRs have been detected in breast cancer patients, but interpretation of their relevance as minimally invasive biomarkers is limited by inconsistent results. Furthermore, the cellular source of circulating miRs and the molecular mechanisms regulating these potential miR biomarkers remain unclear. To address these issues, a highly relevant animal model for in vivo investigation of potential miR biomarkers was established. Aging mice with a Scurfy (sf) mutation of the X-linked tumor suppressor, Foxp3 (Foxp3sf/+), develop spontaneous breast cancer and tumor lung metastases. Analysis of circulating miRs in Foxp3sf/+ female mice revealed that plasma miR-200c/141 and miR-155 levels increase dramatically during tumor progression and lung metastasis, and these results were validated in a small population of breast cancer patients. Thus, these miRs appear as ideal candidates for novel biomarkers to detect the progression of human breast cancer and predict tumor response to therapies. Hypothesis and Goals: The central hypothesis is that circulating miR-200c/141 and miR-155 are useful biomarkers for early detection and prediction of tumor progression and therapeutic response. This hypothesis will be tested in three specific aims: 1) To validate potential miR biomarkers in a large patient population, 2) To determine the utility of the miR biomarkers in the early detection of tumor progression and prediction of therapeutic response, and 3) To elucidate the genesis and regulation of circulating miR biomarkers. Approach: First, the upregulation of the miR biomarkers identified in the Foxp3sf/+ mouse model will be validated in a large population of breast cancer patients using a TaqMan miR assay. The most optimal methods, materials, controls, and matched individuals will be used to ensure the statistical significance of this validation. Second, we will determine if the miR biomarkers are useful for the early detection of tumor progression, especially tumor metastasis. The utility of miR biomarkers in predicting response to treatments in breast cancer patients will be estimated by comparison with current biomarkers. Third, we will use FOXP3 Tet-off MCF7 cells to determine if miRs are released from breast cancer cells and regulated by FOXP3. The Foxp3sf/+ mouse model will be used to further investigate the cellular sources of circulating miR biomarkers. Innovation and Significance: This study will be the first to investigate 1) the utility of the miR biomarkers in predicting response o therapies, and 2) the mechanisms regulating circulating miR biomarkers during tumor progression in vivo. If confirmed, these miR biomarkers will provide non-invasive tools for improving outcomes and decreasing mortality in breast cancers with FOXP3 defects.